Assays which are based on binding between ligands or members of specific binding pairs are widely used to determine the presence and quantities of analytes of interest (e.g., chemical constituents or substances of a sample). Immunoassays, which are a type of ligand-binding assay, are based on the highly specific reaction which occurs between members of specific binding pairs: an antigen and its antibody (e.g., the antibody produced in response to presence of the antigen in a higher animal). In part because of this specificity, immunoassays are particularly useful in detecting analytes which occur in small quantities in a sample.
Agglutination immunoassays are one type of immunoassay and, as their name suggests, the immunochemical reaction which occurs results in clumping of particulates, such as red blood cells or particles to which reagents are bound.
Another often-used type of immunoassay is the radioimmunoassay, in which radioactively-labeled antigens are used as detectable reagents. In this case, the labeled antigen serves as a means of tracing and measuring the distribution of total antigen (labeled and unlabeled) between the antibody-bound and the antibody-free fractions. Quantities of labeled antigen and of antiserum are kept constant and the proportion of counts in the bound fraction is inversely related to the total quantity of antigen present.
Immunoradiometric assays are another form of immunoassay based on the use of radioactively-labeled selected antibodies. They differ from radio-immunoassay procedures in several ways. For example, fewer reactants are generally used, an excess quantity of labeled antibody is added so that all antigen in the standard or sample will be bound, separation of antibody in the bound fraction from antibody in the free fraction is required and there is a direct relationship between counts in the bound fraction and the total quantity of antigen present.
Separation of bound and free-fraction antigen or antibody is generally necessary in immunoassay techniques. For example, in radioimmunoassay procedures, precipitation of the antigen: antibody complex does not occur spontaneously because only small quantities (low concentrations) of reagents are used. Several methods of separating bound from free-fraction reagents have been developed. Pourfarzaneh, M. et al., Methods of Biochemical Analysis, 28:268-295 (1982). These are generally based on differences between the bound and the free fractions (e.g., physical, chemical, physicochemical, immunological differences) and may have limited applicability because, for example, they are time-consuming, complex or unable to effect complete separation of the two fractions.
An immunoassay method which is nonisotopic and makes it possible to separate the bound reagents from free-fraction reagents efficiently and easily would be very useful and would be particularly valuable if it also allowed easy detection and measurement of the separated phases.